Ascorbic acid, or vitamin C, is the most important water-soluble antioxidant in the human circulation. Although humans cannot synthesize ascorbate de novo, it can be efficiently recycled within cells from its oxidized product, dehydroascorbate. Our work has suggested that such ascorbate recycling is particularly important in the human erythrocyte, where such recycling can contribute both to circulating levels of the vitamin, and to protecting the erythrocyte membrane against lipid peroxidative damage. The goals of this proposal are to determine the mechanisms by which erythrocyte ascorbate recycling contributes to the defense against lipid peroxidation in blood, and to establish the extent to which this protection occurs. To achieve these goals, we will utilize techniques of fluorescence spectroscopy, EPR spectroscopy, molecular cloning, and novel biochemical assays. Whether and how ascorbate functions to protect cell membranes and lipoproteins from oxidation is relevant to mechanisms of inflammation, ageing, and atherosclerosis, especially given the fact that dietary supplementation can more than double circulating and tissue levels of the vitamin. In the first specific aim, we will address the question of how reducing equivalents from intracellular ascorbate are transferred across or into the cell membrane. In erythrocytes such transfer appears to be mediated via a transmembrane ascorbate-dependent oxidoreductase enzyme. We will first complete our studies of the kinetic mechanism of this enzyme activity, purify it from human erythrocytes, and clone its cDNA sequence from a bone marrow-derived cDNA library. We will also evaluate the role of ubiquinol-10 (coenzyme Q10) as a cofactor necessary for electron transfer by the enzyme as well as whether the enzyme is functions similarly in a cultured monocyte-derived cell. In the second specific aim-, we will determine whether and to what extent membrane-bound lipid hydroperoxides, alpha-tocopherol and ubiquinone-10 are natural acceptors of electrons derived from ascorbate within erythrocytes. In the third specific aim we will determine whether extracellular ascorbate or its lipophilic derivatives can prevent oxidant-mediated damage in erythrocyte membranes, either directly or through recycling of alpha tocopherol. We will also test the hypothesis that ascorbate-dependent recycling of alpha tocopherol in erythrocytes can replenish depleted stores of alpha tocopherol in human LDL.